Tetracycline repressor-mediated binary regulation system for control of gene expression in transgenic mice

ABSTRACT

The present invention relates to a tetracycline repressor-mediated binary regulation system for the control of gene expression in transgenic mice. It is based, at least in part, on the discovery that, in a transgenic mouse that carries a first transgene under the control of a modified promoter comprising a tetR operator sequence and a second transgene encoding the tetR protein, expression of the first transgene may be efficiently induced by administering tetracycline to the mouse.

This 371 application claims the benefit of PCT/US93/08230, filed Aug.26, 1993, which is a continuation-in-part of U.S. Application Ser. No.07/935,763, filed Aug. 26, 1992, now abandoned.

1. INTRODUCTION

The present invention relates to a tetracycline repressor-mediatedbinary regulation system for the control of gene expression intransgenic animals. It is based, at least in part, on the discoverythat, in a non-human transgenic animal that carries a first transgeneunder the control of a modified promoter comprising a tetR operatorsequence and a second transgene encoding the tetR repressor protein,expression of the first transgene may be efficiently induced byadministering tetracycline to the animal.

2. BACKGROUND OF THE INVENTION 2.1. Control of Gene Expression inTransgenic Animals

The production of transgenic animals for both experiment andagricultural purposes is now well known (Wilmut et al., Jul. 7, 1988,New Scientist pp. 56-59). In research, transgenic animals are a powerfultool that have made significant contributions to our understanding ofmany aspects of biology and have contributed to the development ofanimal models for human diseases (Jaenisch, 1988, Science240:1468-1474). It is also clear that several livestock species can bemade transgenic and these species promise to expand and revolutionizethe method of production and diversity of pharmaceutical productsavailable in the future, in addition to improving the agriculturalqualities of the livestock species (Wilmut et al., supra).

A critical, often neglected, aspect of developing transgenic animals isthe process whereby expression of the newly introduced gene, referred toas the transgene, is controlled. This is an important process sincestringent regulation of transgene expression is often important both forpractical, regulatory and safety reasons and to maintain the health ofthe transgenic animal. In the past either "inducible" or "tissuespecific" regulatory mechanisms have been used. Inducible regulation isdefined herein as a method of gene regulation which allows for some formof outside manipulation of the onset and/or level of transgeneexpression. Tissue specific regulation is defined herein as a method fortargeting transgene expression to particular tissues or organs.

Inducible gene regulation may be achieved using relatively simplepromoter systems such as the metallothionein heat shock promoters, or byusing promoters which are responsive to specific compounds such as theMouse mammary tumor virus LTR which is responsive to glucocorticoidstimulation. More flexible, though more complex inducible regulationsystems can be achieved through a "binary" gene approach which utilizesa transactivator gene product to control expression of a second gene ofinterest. Tissue specific gene regulation usually consists of simplesingle gene methods (Byrne et al., 1989, Proc. Natl. Acad. Sci. U.S.A.86:5473-5477; Ornitz et al., 1991, Proc. Natl. Acad. Sci. U.S.A.88:698-702), although binary transactivator systems can also provide ahigh degree of tissue specificity.

These current systems provide only a limited ability to control the timeof transgene expression within individual animals. In this respecttissue specific promoter elements provide no method to control the onsetof transgene activity, but function merely to target gene expression todefined sites. Simple inducible promoters such as metallothioneingenerally lack tissue specificity and usually have some aspect ofendogenous basal expression which cannot be controlled. Thus even forthe extensively used inducible metallothionein promoter this approach atbest only permits selection of the time at which a relative increase intransgene expression can be induced.

Binary transactivation systems typically consist of two transgenicanimals. One animal contains the gene of interest controlled by apromoter element that requires a specific transactivator gene productfor expression. Thus, the gene of interest is not expressed in theabsence of the transactivator. A second transgenic animal is then madewhich expresses the required transactivator in the desired tissue. Bymating these two transgenic animals, offspring containing both the geneof interest and the transactivator transgene can be produced. Only inthese doubly transgenic animals is the gene of interest expressed. Sinceexpression of the gene of interest requires the transactivator, thisbinary approach dramatically reduces or eliminates any undesirable basalexpression inherent in simple inducible systems. Additionally, ifexpression of the transactivator is targeted using a tissue specificpromoter, then in the double transgenics, expression of the gene ofinterest is in effect targeted to the same specific tissue. Binarysystems provide therefore a low resolution method of temporal regulationin as much as they allow the determination of which generation ofanimals will express the gene of interest. These systems provide littleability, however, to control the time and level of gene expressionwithin an individual transgenic animal.

For many applications it is necessary to accurately control the time andpattern of transgene expression within an individual transgenic animal.For example, many attempts have been made to produce transgenic pigswhich express increased levels of growth hormone (Vize et al., 1988, J.Cell Sci. 90:295-300; Pinkert et al., 1990, Dom. Animal Endocrinol.7:1-18). Elevated growth hormone levels dramatically decrease the amountof body fat in pigs, and increase the animals overall feed efficiency.These effects would be beneficial, both to the consumer who couldpurchase a leaner, healthier product, and to the producer who can profitfrom having a more efficient animal. To date however, all attempts toincrease the level of growth hormone through production of transgenicpigs have also produced serious pathological conditions which greatlyreduce the health of the animals. These pathologies are the directresult of uncontrolled, constitutive expression of growth hormone, sincemany studies using exogenous hormone administration for short periods oftime have not produced pathologies, while still benefiting feedefficiency and fat content. In this situation, a regulatory method tocontrol onset and level of expression from a growth hormone transgenewould be extremely useful.

2.2. Repressor-Mediated Gene Control

Transcriptional repressors are usually allosteric DNA binding proteinswith at least two functional sites. One site on the protein is used tobind DNA. The DNA binding site binds to a defined DNA sequence which isknown as the operator site. Operator sites usually consist ofpalindromic sequences of 12 or more base pairs. A gene which isregulated by a repressor must have at least one operator site locatedwithin its promoter/regulatory region. A second site on the repressorprotein binds a specific ligand, usually a small macromolecule such asan amino acid, sugar, or antibiotic. When the ligand is bound to therepressor, it causes a conformational shift such that the affinity ofthe repressor for the operator sequence is greatly reduced. For thisreason, the ligand is frequently referred to as the "inducer", since itcauses the repressor to disassociate from the operator, therebyeliminating the repressor's effect and allowing expression of the gene.

Only the bacterial repressors Lacl, LexA and tetR have been shown tofunction in mammalian (Lacl and LexA) or plant (tetR) tissue culturecells. The first report of utilizing bacterial repressors in eukaryoteswas from Brent and Ptashne who showed that LexA could function in yeast(1984, Nature 312:612-615). Subsequently, both LexA and Lacl have beenshown to function in mammalian tissue culture systems (Smith et al.,1988, EMBO J. 7:3975-3981). Of these repressors Lacl has been mostextensively studied. For Lacl repression, single or multiple operatorsites have been positioned in three major locations: (i) between thetranscription start site and the first codon of the mRNA; (ii) betweenthe TATA-box sequence and the transcription start site; and (iii)between the TATA-box sequence and any more distal regulatory signalsequences. These studies reveal two predominant results. First,operators located in all three positions were effective in rendering themodified promoter subject to Lacl repression. Second, the presence ofmultiple operator sequences allowed greater levels of repression thandid single operator insertions. From these studies it appears the Laclrepressor causes repression of mammalian promoters through two basicmechanisms. If the operators are located downstream of the transcriptionstart site, Lacl appears to block expression by inhibiting mRNAelongation. That is to say, the LacI repressor blocks the progress ofRNA polymerase by steric interference. When operator sequences arelocated in other positions, LacI seems to inhibit protein-proteininteractions between the cellular factors normally involved intranscription initiation.

Gatz and Quail (1988, Proc. Natl. Acad. Sci. U.S.A. 85:1394-1397) havedemonstrated tetR function in a plant protoplast culture system. Plantprotoplasts were transfected with a tetR gene expressed from acauliflower mosaic virus (CAMV) promoter along with a CAT reporter gene,regulated by a modified CAMV promoter. In contrast to the results withLacl, Gatz and Quail showed that tetR operators positioned between thetranscription start site and the first codon of the CAT mRNA were notresponsive to tetR repression. Therefore the tetR protein does notappear to be able to block the procession of RNA polymerase. Effectiverepression by tetR was only observed when the operator sequence waspositioned such that the CAMV TATA-box element was flanked by the two 19palindromes of the tetR operator. With this modification, effectiverepression of the reporter gene, and induction with tetracycline couldbe achieved. This suggests that repression by tetR specifically inhibitsthe initiation of transcription, in this case apparently by blocking thebinding of the TATA-box binding factors.

Recently the tetR system has been shown to function in transgenicplants. Gatz et al. (1991, Mol. Gen. Genet. 227:229-237) have introducedtheir original tetR responsive CAMV promoter, in which the operatorsites flank the TATA-box into transgenic tobacco plants. Unexpectedly,this promoter, which exhibited very good regulation in tissue cultureassays was not very effective in regulating gene expression intransgenic plants. Instead they found that effective repression andinduction in transgenic plants occurred when the operator sites werepositioned just downstream of the normal transcription start site.

3. SUMMARY OF THE INVENTION

The present invention relates to a tetracycline repressor-mediatedbinary regulation system for the control of gene expression in non-humantransgenic animals. It is based, at least in part, on the discovery thatin transgenic mice carrying two transgenes, the first encoding bovinegrowth hormone (bGH) under the control of a PEPCK promoter modified tocomprise the tetR operator sequence at the NheI site, and the secondencoding tetR repressor protein under the control of an unmodified PEPCKpromoter, expression of bGH could be efficiently and selectively inducedby administering tetracycline to the transgenic mice.

In particular embodiments, the present invention provides for (i) animalpromoter elements modified to comprise a tetR operator sequence; (ii)nucleic acid molecules comprising a gene of interest under the controlof such a modified promoter; (iii) non-human transgenic animals thatcarry a transgene under the control of said modified promoter and/or atransgene encoding the tetR repressor protein; and (iv) a method ofselectively inducing the expression of a gene of interest in a non-humantransgenic animal comprising administering tetracycline to a non-humantransgenic animal that carries a first transgene, which is the gene ofinterest under the control of a promoter modified to comprise a tetRoperator sequence and a second transgene encoding the tetR repressorprotein.

The present invention offers the advantage that, in the absence oftetracycline, expression of the gene of interest occurs at only very lowlevels due to efficient repression by tetR. In preferred, non-limitingembodiments of the invention, repression by tetR is further enhanced byutilizing a synthetic tetR gene which is devoid of splice signals andhas optimized codon usage for mammalian cells. Accordingly, the presentinvention allows tight control of gene expression in transgenic animalsby withholding or administering tetracycline.

4. DESCRIPTION OF THE FIGURES

FIG. 1. A. Nucleotide sequence of tetR operator as it occurs in Tn10(SEQ ID NO:1), and in the oligonucleotides used to produce the modifiedPEPCK promoter elements (SEQ ID NO:2). Bold face lettering represent theOP1 and OP2 tetR binding sites. The general purpose oligonucleotide (SEQID NO:2) is the sequence from p∂∂7. The flanking EcoRI and AccIrestriction sites used to excise this operator sequence are indicated.Additional restriction sites present in the plasmid, but not indicatedhere, which can be used to excise the operator (SEQ ID NO:3) includePstI, BamHI, SpeI, Sbal, NotI, EagI, SacII, BstXI, and SacI on the 5'side and XhoI, ApaI and KpnI on the 3' side. The sequence of thePEPCK-TATA box operator is also indicated (see methods)(SEQ ID NO:3).

FIG. 1. B. Nucleotide sequence of the ∂∂7 operator (SEQ ID NO:4). Lowercase letters correspond to polylinker sequence. The 5' EcoRI and 3' AccIrestriction sites used for producing the modified PEPCK promoters (Pck₋₋A and Pck-N) are indicated. The 10 base pair linker beween OP1 and OP2is underlined. Additional polylinker restriction sites available in p∂∂7include PstI, BamHI, SpeI, XbaI, NotI, EagI, SacII, BstXI, and SacI onthe 5' side and XhoI, ApaI and KpnI on the 3' side.

FIG. 2. A representation of the three modified PEPCK promoter elements.Construct 251 has the ∂∂7 operator sequence integrated in the AccI siteof PEPCK, just 5' of the TATA-box control element. Construct 252 has the∂∂7 operator sequence incorporated into the NheI site of PEPCK, just 3'of the TATA-box element. Construct 261 incorporates the TATA-specificoperator sequence which is integrated between the 5' AccI site and the3' NheI sites.

FIG. 3. Structure of the modified PEPCK controlled bovine growth hormonegenes. The Pck₋₋ AbGH and Pck₋₋ NbGH genes differ only in the site ofoperator insertion. For Pck₋₋ AbGH the operator is inserted at the AccIsite 5' of the PEPCK TATA-box element. For Pck₋₋ NbGH the operator isinserted into the NheI site 3' of the TATA-box element (pPCK₋₋ NbGH hasbeen deposited with the ATCC and assigned accession No: 69400). In thePck₋₋ TbGH gene, a TATA-box specific oligonucleotide was used, and thissequence was inserted between both the AccI and NheI sites. A. Indicatedthe probe used for S1 hybridization.

FIG. 4. S1 Nuclease protection assay to map the 5' start site of bGHfrom the Pck₋₋ N promoter. Total liver RNA (10 μg) was hybridized to a280 bp 5' labelled probe from the Pck₋₋ NbGH gene in 40 mM PIPES(Ph6.4), 1 Mm EDTA, 400 mM NaCl, 80% formamide at 55° overnight. Theprobe spanned from the HinfI site in the 5' untranslated leader sequenceof bGH to the PvuII site 5' of the TATA-17 box. The probe includes thetet-operator sequence of Pck₋₋ N (see FIG. 3). After hybridization 300μl of ice cold digestion buffer (280 mM NacL, 50 Mm SODIUM ACETATE(Ph4.5), 4.5Mm ZnSO₄, 20 μg/ml carrier DNA and 500 units S1 nuclease)was added and incubated at 37° for 30 minutes. The reaction as stoppedby adding 80 μl of Stop Buffer (4M Ammonium acetate, 50 mM EDTA and 50μg/ml tRNA), extracted with phenol/chloroform, precipitated with ethanoland analyzed on a 6% sequencing gel. The arrow indicates the protectedfragment. Initiation of bGH mRNA from the modified Pck₋₋ N promoteroccurs approximately 20 bp 3' of the TATA-box. This initiation siteplaces the start of the message just prior to the first tetR bindingsite. This result indicates that the bGH mRNA starts from a single capsite, and suggests that tetR repression is due to a block intranscription initiation. Furthermore, unrepressed bGH expressionappears to be due to limited tetR expression.

FIG. 5. Nucleotide sequence (SEQ ID NO:5) of the tetR repressor protein(SEQ ID NO:6) gene.

FIG. 6. Alterative, nonlimiting promoters of interest (Chick β-actin:SEQID NO:7; Mouse Albumin:SEQ ID NO:8; Human CD-2:SEQ ID NO:9; Humanalpha-globin:SEQ ID NO:10; Mouse Cardiac Myosin Heavy Chain:SEQ IDNO:11). Asterisks indicate sites at which tetR operator sequence may beinserted.

FIG. 7. Northern blot analysis of bGH mRNA in liver of F1 generationanimals.

FIG. 8. Northern blot analysis of bGH mRNA expression in four transgeniclines.

FIG. 9A. Tissue specificity of bGH expression in Line 10-2 in thepresence of 50 μg/ml tetracycline. Northern blot analysis of bGHinduction in a variety of tissues. Only the liver and kidney showsignificant expression.

FIG. 9B. Tetracycline induction of bGH in Line 10-2. Both liver andkidney, which are the only sites for bGH expression in FIG. 9A, alsoshow tetracycline dependent bGH expression.

FIG. 10. 345 Repressor Construct.

FIG. 11. Induction of bGH expression in Construct 345 Offspring.Northern blot analysis of liver RNA from F1 animals containing the 345construct. Only animals from line 14 exhibit tetracycline dependent bGHexpression.

FIG. 12. Expression and alternative processing of tetR transgene. ARNase protection probe which extends from the Nrul site of tetR 3' tothe end of the gene was used. This probe includes only tetR codingsequences and should give a fully protected fragment of approximately400 base pairs. A protected fragment of approximately 220-260 base pairsis observed, which is far smaller then predicted.

FIG. 13. 5' Structure of tetR mRNA. Liver RNA was treated with reversetranscriptase and amplified by PCR. The RNA was amplified using twodifferent pairs of primers. The first primer pair (TZ-1 and TZ-4) shouldproduce a 619 base pair product. The second primer pair (TZ03 and TZ04)should produce a 498 base pair product. The sequence of the primers are:

TZ-1(SEQ ID No:21):5'CCGCATATGATCAATTCAAGGCCGAATAAG3'

TZ-3(SEQ ID No:22):5'CTTTAGCGACTTGATGCTCTTGATCTTCCA3'

TZ-4(SEQ ID No:23):5'AATTCGCCAGCCATGCCAAAAAAGAAGAGG3'

The TZ-4 primer is common to both primer pairs and is the 5' primerwhich encompasses the start codon of the tetR and mRNA. Primer TZ-1 andTZ-3 are two different 3' primers both of which are in the tetR codingregion. When amplified, these primer pairs produced smaller thenexpected products (approx. 215 bp vs. 619 bp for TZ-4 and TZ-1, andapprox. 94 bp vs. 498 bp for TZ-4 and TZ-3). The products of thisreaction were cloned and sequenced. Sequencing revealed the presence ofan unexpected intron which spanned from near the Xbal site at the startof tetR to a splice acceptor just 8 base pairs 5' of the TZ-3 primer.

FIG. 14. Composition analysis of Wild Type Tn10 tetR gene. The Tn10 tetRcoding sequence was analyzed on a desktop computer using Mac Vectorsoftware. The figure shows a diagram of the tetR coding region with theplus strand splice doner (D) and splice acceptor (A) signal sequencesindicated. For reference the location of the XbaI restriction is alsoindicated. The first graph depicts the percentage of G and C bases inthe coding region of tetR. There are several domains of very low GCcontent. The bottom graph is an analysis of codon bias. The dark line isa comparison of the tetR codon usage to a mouse codon bias table. Valueslower than 1.0 are indicative of sequences which may translate poorly.For reference, a comparison of tetR to a Tobacco codon bias table isincluded (light line). In transgenic tobacco, the tetR regulation systemfunctions very efficiently, suggesting that for this gene, codon biasmay be an important factor for efficient expression.

FIG. 15. Synthetic tetR Component Sequences (LT-1:SEQ ID NO:16; LT-2:SEQID NO:17; LT-3:SEQ ID NO:18; LT-5:SEQ ID NO:19). The components of thesynthetic tetR gene were synthesized by Midland Laboratories as fouroverlapping double stranded DNA cassettes. The sequence of thesecassettes are shown. Each cassette was blunt cloned into the Hinc2 siteof pUC19 and sequenced to verify authenticity. The resulting plasmidspLT1, pLT2, pLT3 and pLT5 can be used as the source material to assemblethe entire synthetic tetR coding sequence since each contains anoverlapping unique restriction site (bold face) through which they canbe joined.

FIG. 16. Sequence of Synthetic tetR gene (SEQ ID NO:20).

FIG. 17. Composition analysis of synthetic tetR. These graphs wereproduced using the same software described in FIG. 15. The figuredepicts the structure of the synthetic tetR gene, now devoid of splicedonor signal sequences, with only a single splice acceptor signalremaining (A). This is not the splice acceptor which was active in the345 construct. The percentage of G and C bases has been significantlyimproved, while the frequency of CpG base pairs has been kept to aminimum. A CpG base pair is frequently the site for DNA methylation,which can negatively effect the expression of a gene. The codon bias ofthe synthetic tetR gene is also vastly improved. The graph depicts theresults when the synthetic tetR coding sequence is compared to the samemouse codon bias table used previously.

5. DETAILED DESCRIPTION OF THE INVENTION

For purposes of clarity of description, and not by way of limitation,the detailed description of the invention is divided into the followingsubsections:

(i) the tetR operator;

(ii) modified promoters containing the tetR operator; and

(iii) utility of the invention.

5.1. The TetR Opreator

In order to practice the instant invention, the tetR operator sequenceis inserted into a suitable animal promoter sequence in order to renderthat promoter subject to control by tetR repressor protein. A diagram ofthe tetR operator sequence is depicted in FIG. 1.

It may be convenient to clone the tetR operator into a vector, such as aplasmid or a phage, to facilitate its propagation. Cloned operatorsequence may then be rendered available for insertion into a promoter ofinterest, as set forth in Section 5.2., infra.

In a particular, nonlimiting embodiment of the invention, tetR operatorsequence may be cloned as follows: Four oligonucleotides, which whenannealed produce the two 19 bp OP1 and OP2 palindromic sequences of thetetR operator may be synthesized; the sequences of said oligonucleotidesare as follows:

X-1 (SEQ ID NO:12). 5'ACTCTATCATTGATAGAGT3'

X-2 (SEQ ID NO:13). 5'ACTCTATCAATGATAGAGT3'

X-3 (SEQ ID NO:14). 5'TCCCTATCAGTGATAGAGA3'

X-4. 5'TCTCTATCACTGATAGGGA3'

Oligonucleotides X-1 and X-2 are complementary and, when annealed, formthe OP1 operator. Similarly, oligonucleotides X-3 and X-4, whenannealed, produce the OP2 operator site. The OP1 oligonucleotides maythen be directly cloned into the EcoRV site of the Bluescript(Stratagene) polylinker to form plasmid X. OP2 oligonucleotides may thenbe cloned into a Mung bean nuclease blunted ClaI site of plasmid X toform plasmid Y. The resulting tetR operator may then be propagated andthen excised from plasmid Y as an EcoRI, AccI fragment which may beend-filled with T4 polymerase and gel purified.

It is preferable that the separation between OP1 and OP2 is about 10-11bp.

Analogous methods may be used to insert the tetR operator site intoother suitable vectors.

5.2. Modified Promoters Containing the tetR Operator

According to the invention, the tetR operator may be inserted into asuitable animal promoter so as to render that promoter subject torepression by tetR repressor protein. Any animal promoter maybe used;strategies for promoter selection are set forth in Section 5.3., infra.

In preferred embodiments of the invention,the tetR operator sequence ispositioned 3' to the TATA-box sequence. A nonlimiting list of promoterswhich may be used according to the invention is set forth in FIG. 6,together with the proximal portion of the promoter in the vicinity ofthe TATA-box, which is underlined.

In a specific, nonlimiting embodiment of the invention, the tetRoperator site may be inserted into the NheI site of the PEPCK promoter(Wynshaw-Boris et al., 1984, J. Biol. Chem. 259:12161-12169). A diagramof the PEPCK promoter containing the tetR operator sequence of the NheIsite is presented in FIG. 2. For insertion of the operator sequence, thePEPCK promoter may be cut with NheI and end-filled with T4 polymerase;tetR opera tor, prepared as set forth in Section 5.1., suara, may thenbe blunt-ligated into place.

5.3. Utility of the Invention

5.3.1. STRATEGY

The strategy of the invention is to prepare a non-human transgenicanimal that comprises two transgenes. The first transgene, termed "A,"is a gene of interest, the expression of which is desirably controlled.Virtually any gene of interest may be. used, including, but not limitedto, growth hormone, hemoglobin, low density lipoprotein receptor,insulin, genes set forth in Table I, etc.

                  TABLE 1    ______________________________________    Other Genes Of Interest    Gene                Disease/Affect    ______________________________________    ADA Adenosine deaminase                        Immuno-deficiency    TNF Tumor necrosis factor                        Anti-cancer    IL-2 Interleukin-2  Anti-cancer    LDL low density     hypercholesterolemia    Factor IX           hemophelia    Factor VIII         hemophelia    β-glucosidase  Gauchers disease    CFTR Cystic fibrosis                        Cystic fibrosis    transmembrane regulator    HPRT Hypoxanthine-quanine                        Lesch-Nyhan syndrome    phosphoribosyltransferase    UDP-glucuronyl transferase                        Crigler-Najjar syndrome    Growth Hormone receptor                        Growth    Insulin-like growth factor                        Growth    Growth hormone releasing                        Growth    factor    ______________________________________

The expression of gene "A" is under the transcriptional control ofpromoter "B". Promoter B comprises a tetR operator sequence, asdiscussed supra. Promoter B desirably defines the time and tissue windowin which the transgene may be induced; for example, promoter A may be atissue specific promoter such as the PEPCK promoter (which is expressedselectively in liver and becomes active shortly prior to birth). Thesecond transgene encodes the tetR repressor, the sequence of which isset forth in FIG. 5.

Analysis of the Tn10 tetR coding sequence indicates that the codon usagefor this gene is poorly suited for expression in mammalian cells (FIG.15). To optimize tetR expression in mammalian cells a new tetR repressorgene was designed (See, Section 7, infra), which may be utilized inalternative embodiments of the invention. The synthetic tetR gene(syn-tetR) is designed to encode exactly the same protein product as thebacterial Tn10 tetR gene but optimizes codon usage for mammalian cells.The percentage of G and C bases has been significantly improved, whilethe frequency of CpG base pairs has been minimized. A CpG base pair isfrequently the site for DNA methylation which can negatively affect theexpression of a gene. In addition, the syn-tetR gene is devoid of anysplice signals, decreasing the likelihood of aberrant splicing of theRNA which may result in production of a non-functional message. Thesequence of the synthetic tetR gene is depicted in FIG. 16. Plasmidscomprising these sequences may be constructed using plasmids pLT-1,pLT-2, pLT-3 and pLT-5 (deposited with the American Type, CultureCollection (ATCC) and assigned accession numbers 69396, 69397, 69398,and 69399, as described in Section 7, infra.

In further embodiments, the present invention provides for additionalsynthetic tetR genes from which one or more splice sites have beendeleted or for which codon usage has been further optimized.

The present invention covers synthetic tetR genes having the sequenceset forth in FIG. 16 and for functionally equivalent variants of thatsequence.

In specific, non-limiting embodiments of the invention, a nuclearlocalization signal may be added to a natural or synthetic tetR gene tofacilitate its expression (See, Section 7, infra).

Expression of tetR is controlled by promoter "C". While it is preferablethat promoter C be the same as promoter B except that promoter C doesnot contain a tetR operator sequence, any promoter which providesexpression of tetR so as to repress expression of gene "A" during theperiod when it is desirable to repress expression of "A" may be used.

For example, and not by way of limitation, a transgenic animal may beproduced which carries a first transgene which is bovine growth hormoneunder the control of a PEPCK promoter modified to contain a tetRoperator sequence at the NheI site and a second transgene which is tetRrepressor protein under the control of an unmodified PEPCK promoter; seeSection 6, infra. The pPCK₋₋ NbGH construct has been deposited with theATCC and assigned accession number 69400.

5.3.2. TRANSGENIC ANIMALS OF THE INVENTION

The binary repressor system of the invention may be used to control geneexpression in any non-human transgenic animal, including, but notlimited to, transgenic mice, pigs, goats, cows, rabbits, sheep, etc. Thepresent invention provides for such non-human transgenic animals carringas transgenes nucleic acid constructs described herein, includingnatural or synthetic tetR repressor proteins and operator sequences.

Transgenes may be introduced by microinjection, transfection,transduction, electroporation, cell gun, embryonic stem cell fusion, orany other method known in the art. The transgenes of the invention maybe co-introduced into a single animal or may be introduced into twoindividual animals that are subsequently mated to produce doublytransgenic offspring.

For example, for the production of transgenic mice, the followinggeneral protocol may be used. Male and female mice are mated atmidnight. Twelve hours later, the female may be sacrificed and thefertilized eggs may be removed from the uterine tubes. Foreign DNA maythen be microinjected (100-1000 molecules per egg) into a pronucleus.Shortly thereafter, fusion of the pronuclei (a pronucleus or the malepronucleus) occurs, and, in some cases, foreign DNA inserts into(usually) one chromosome of the fertilized egg or zygote. The zygote maythen be implanted into a pseudo-pregnant female mouse (previously matedwith a vasectomized male) where the embryo develops for the fullgestation period of 20-21 days. The surrogate mother then delivers themice and by four weeks transgenic pups may be weaned from the mother.

According to another embodiment of the invention, a transgenic pig maybe produced, briefly, as follows. Estrus may be synchronized in sexuallymature gilts (>7 months of age) by feeding an orally active progestogen(e.g. allyl trenbolone, AT: 15 mg/gilt/day) for 12 to 14 days. On thelast day of AT feeding all gilts may be given an intramuscular injectionof prostaglandin F_(2a) (Lutalyse: 10 mg/injection) at 0800 and 1600hours. Twenty-four hours after the last day of AT consumption all donorgilts may be administered a single intramuscular injection of pregnantmare serum gonadotrophin (1500 U). Human chorionic gonadotrophin (750IU) may be administered to all donors at 80 hours after pregnant mareserum gonadotrophin.

Following AT withdrawal, donor and recipient gilts may be checked twicedaily for signs of estrus using a mature boar. Donors which exhibitedestrus within 36 hours following human chorionic gonadotrophinadministration may be bred at 12 and 24 hours after the onset of estrususing artificial and natural (respectively) insemination.

Between 59 and 66 hours after the administration of HCG one- andtwo-cell ova may be surgically recovered from bred donors using thefollowing procedure. General anesthesia may be induced by administering0.5 mg of acepromazine/kg of bodyweight and 1.3 mg of ketamine/kg via aperipheral ear vein. Following anesthetization, the reproductive tractmay be exteriorized following a mid-ventral laparotomy. A drawn glasscannula (O.D. 5 mm, length 8 cm) may be inserted into the ostium of theoviduct and anchored to the infundibulum using a single silk (2-0)suture. Ova may then be flushed in retrograde fashion by inserting a 20g needle into the lumen of the oviduct 2 cm anterior to the uterotubaljunction. Sterile Dulbecco's phosphate buffered saline (PBS)supplemented with 0.4% bovine serum albumin (BSA) may be infused intothe oviduct and flushed toward the glass cannula. The medium may becollected into sterile 17×100 mm polystyrene tubes. Flushings may betransferred to 10×60 mm petri dishes and searched at a lower power (50×)using a Wild M3 stereomicroscope. All one- and two- cell ova may bewashed twice in Brinster's Modified Ova Culture -3 medium (BMOC -3)supplemented with 1.5% BSA and transferred to 50 μl drops of BMOC-3medium under oil. Ova may be stored at 38° C. under a 90% N_(z), 5%O_(z). 5% Co₂ atmosphere until microinjection is performed. One andtwo-cell ova may be placed in an Eppendorf tube (15 ova per tube)containing 1 ml HEPES medium supplemented wit 1.5% BSA and centrifugedfor 6 minutes at 14,000 g in order to visualize pronuclei in one-celland nuclei in two-cell ova. Ova may then be transferred to a 5-10 μldrop of HEPES medium under oil on a depression slide. Microinjection maybe performed using a Laborlux microscope with Nomarski optics and twoLeitz micromanipulators. 10-1700 molecules of construct DNA (linearizedat a concentration of about 1 ng/μl of Tris-EDTA buffer) may be injectedinto one pronucleus in one-cell ova or both nuclei in two-cell ova.Microinjected ova may be returned to microdrops of BMOC-3 medium underoil and maintained at 38° C. under a 90% N₂, 5% CO₂, 5% O₂ atmosphereprior to their transfer to suitable recipients. Ova may preferably betransferred within 10 hours of recovery. Only recipients which exhibitestrus on the same day or 24 hours later than the donors may preferablybe utilized for embryo transfer. Recipients may be anesthetized asdescribed supra. Following exteriorization of one oviduct, at least 30injected one- and/or two-cell ova and 4-6 control ova may be transferredin the following manner. The tubing from a 21 g×3/4 butterfly infusionset may be connected to a 1 cc syringe. The ova and one to two mls ofBMOC-3 medium may be aspirated into the tubing. The tubing may then befed through the ostium of the oviduct until the tip reaches the lowerthird or isthmus of the oviduct. The ova may be subsequently expelled asthe tubing is slowly withdrawn. The exposed portion of the reproductivetract may be bathed in a sterile 10% glycerol 0.9% saline solution andreturned to the body cavity. The connective tissue encompassing thelinea alba, the fat, and the skin may be sutured as three separatelayers. An uninterrupted Halstead stitch may be used to close the lineaalba. The fat and skin may be closed using a simple continuous andmattress stitch, respectively. A topical antibacterial agent (e.g.Furazolidone) may then be administered to the incision area. Recipientsmay be penned in groups of about four and fed 1.8 kg of a standard 16%crude protein corn-soybean pelleted ration. Beginning on day 18 (day0=onset of estrus), all recipients may be checked daily for signs ofestrus using a mature boar. On day 35, pregnancy detection may beperformed using ultrasound. On day 107 of gestation recipients may betransferred to the farrowing suite. In order to ensure attendance atfarrowing time, farrowing may be induced by the administration ofprostaglandin F_(2a) (10 mg/injection) at 0800 and 1400 hours on day 112of gestation. In all cases, recipients may be expected to farrow with 34hours following PGF 2a administration.

As used herein, the term "transgenic animal" refers to animals thatcarry a transgene in at least some of their somatic cells, andpreferably in at least some of their germ cells.

5.3.3. INDUCTION

Induction of expression of the gene of interest in transgenic animals ofthe invention may be achieved by administering, to the animal, acompound that binds to tetR so that tetR repressor function isinhibited. Examples of such compounds include tetracycline andtetracycline-like compounds, including, but not limited to, apicycline,chlortetracycline, clomocycline, demeclocyline, guamecycline,lymecycline, meclocycline, methacycline, minocycline, oxytetracycline,penimepicycline, pipacycline, rolitetracycline, sancycline, andsenociclin.

Administration of the inducer can be through direct injection, water,feed, aerosol, or topical application. The choice of method will dependon the promoters used and the specific application of the transgenicanimals. For example, injection, water and feed would provide inducer toall of the animals tissues. In our case, administration through water orfeed would be the preferred method to control growth hormone expressionin transgenic pigs. Aerosol spray could be used to attain highantibiotic concentrations in the lung. This may be appropriate forexample in a cystic fibrosis or emphysema model. Topical application tothe skin is also possible and could be used in models of acne, hairloss, wound healing or viral infection.

Induction of the gene of interest is accomplished by administering aneffective amount of inducer, as described above. An effective amount ofinducer may be construed to mean that amount which increases expressionof the gene of interest by at least about 50 percent. As the LD₅₀ fortetracycline HCl in rats is about 6643 mg/kg and the therapeutic dose isbetween about 25-50 mg/kg, an effective dose of tetracycline, asinducer, is between about 5-50 mg/kg and preferably between about 5-15mg/kg.

6. EXAMPLE: TETRACYCLINE REPRESSOR-MEDIATED BINARY REGULATION SYSTEM FORCONTROL OF BOVINE GROWTH HORMONE EXPRESSION IN TRANSGENIC MICE 6.1.Materials and Methods

6.1.1. CONSTRUCTION OF PLASMIDS

Plasmid p∂∂7 contains a functional tetR operator site cloned within aBluescript (Stratagene) polylinker. This plasmid is useful forpropagating the operator sequence, and as a source of operator sites forinsertion into the PEPCK promoter or any other promoter element. Thep∂∂7 plasmid was made as follows. Four oligonucleotides, which whenannealed produce the two 19 bp OP1 and OP2 palindromic sequences of thetetR operator were synthesized. The sequences of each oligonucleotide islisted below.

X-1.5' ACTCTATCATTGATAGAGT 3' (SEQ ID NO:12)

X-2.5' ACTCTATCAATGATAGAGT 3' (SEQ ID NO:13)

X-3.5' TCCCTATCAGTGATAGAGA 3' (SEQ ID NO:14)

X-4.5' TCTCTATCACTGATAGGGA 3' (SEQ ID NO:15)

Oligonucleotides X-1 and X-2 are complementary and when annealed formthe OP1 operator. Similarly oligonucleotides X-3 and X-4 produce the OP2operator site. The OP1 oligonucleotides were directly cloned into theEcoRV site of the Bluescript polylinker. The resulting plasmid pSOPI wassequenced to verify the integrity of the insert. OP2 oligonucleotideswere subsequently cloned into a Mung bean nuclease blunted Clal site ofpSOPI to produce p∂∂7. Due to a cloning artifact produced by the Mungbean nuclease, the operator in p∂∂7 consists of the two 19 bp OP1 andOP2 sequences separated by linker of only 10 base pairs. This differencedoes not effect tetR binding. The sequence of the p∂∂7 operator site isshown in FIG. 1B. The 55 base pair tetR operator was excised from p∂∂7as an EcoRl, AccI fragment, end filled with T4 polymerase, and gelpurified. This fragment was subsequently used to produce the modifiedPEPCK promoters Pck₋₋ N and Pck₋₋ A.

Plasmids Pck₋₋ A and Pck₋₋ N were produced by inserting the 55 bp tetRoperator into the unique AccI and NheI sites (respectively) of the PEPCKpromoter (pPCK₋₋ NbGH has been deposited with ATTC and assignedaccession No: 69400). For both plasmids the promoter was cut with theappropriate restriction enzyme, end filled with T4 polymerase and thetetR operator blunt ligated into place. A third modified PEPCK promoter,Pck₋₋ T was produced in which the OP1 and OP2 operator sequences werepositioned to flank the PEPCK TATA-box element. To produce Pck₋₋ T a newoligonucleotide (5'ACTCTATCATTGATAGAGTTACTATTTAAATCCCTATCAGTGATAGAGA3')(SEQ ID NO:13)) was produced. Thisoligonucleotide was kinased with T4 polynucleotide kinase and annealedto kinased X-2 and X-4 which are complementary to the first and last 19bp. The complete double stranded 49 bp operator was produced by fillingin the 11 bp linker region, which includes the PEPCK TATA-box element,with Klenow. The final product was then blunt cloned into an AccI, NheIcut PEPCK promoter. All three modified promoters were sequenced toverify the inserts. FIG. 2 depicts the structure of these promoters.

6.1.2. REPRESSOR CONSTRUCT

Plasmid pBI501 contains a 701 bp HincII fragment from E. coli Tn10,cloned into the HincII site of pUC8. The HincII insert contains theentire tetR coding sequence along with 21 bp of 5' and 55 bp of 3'untranslated DNA. This insert was excised from the parent plasmid andsubcloned into a plasmid with a more suitable polylinker to producepSTET7. To this plasmid a 870 bp XhoI, BamHI fragment derived from PMSG(Pharmacia), containing the SV40 small-T intron and polyadenylationsignal sequences was inserted at the HindII site 3' of the tetR codingregion to produce pSTetRSv. Finally an unmodified 610 bp PEPCK promoterwas inserted at the EcoRl site of pSTETRSv to produce pPck₋₋ tetRSv. ThePEPCK promoter is identical to the promoter used to produce pPck₋₋ A,pPck₋₋ N, and pPck₋₋ T except that it does not contain a tetR operatorsite. This PEPCK promoter has been previously used in transgenic animalsand is known to target gene expression specifically to the liver.

6.1.3. GROWTH HORMONE GENES

Plasmid pGH-SAF107 contains a 2.2 kb BamHI, EcoRI genomic fragment ofthe bovine growth hormone (bGH) gene, blunt ligated into an EcoRV site.To this vector each of the modified PEPCK promoters was added by bluntligating the promoter into the BamHl site of pGH-SAF107. The structureof the resulting plasmids is depicted in FIG. 3. Plasmid pPCK₋₋ NbGH wasdeposited with the ATCC and assigned accession number 69400. Forproduction of transgenic animals, each of the PEPCK-bGH genes wasexcised from the vector using Xhol and Sacl, gel fractionated andpurified using an Elutip column.

6.1.4. TRANSGENIC MICE

Transgenic mice were made which contain both the Pck₋₋ tetRSv gene andone of the modified PEPCK promoters controlling bGH. Table 2 lists thenumber of eggs injected, offspring produced and number of transgenicsderived for each construct.

                  TAELE 2    ______________________________________                   Eggs    Eggs     Live    Construct      injected                           transferred                                    Born Transgenic    ______________________________________    Pck.sub.-- AbGH + Pck.sub.-- tetRSv                   233     194      40   14 (0.35)    (251)    Pck.sub.-- NbGH + Pck.sub.-- tetRSv                   268     208      30    9 (0.3)    (252)    Pck.sub.-- TbGH + Pck.sub.-- tetRSv                   227     197      25    5 (0.2)    (261)    ______________________________________

6.2. Results and Discussion

Once the transgenic founder animals were identified, they were weighedeach week. Table 3 lists the mean weights of each group of transgenicanimal at 11 weeks of age.

                  TABLE 3    ______________________________________    Construct          Sex      Weight    ______________________________________    Pck.sub.-- AbGH + Pck.sub.-- tetRSv (9)                       male     36.122 (12.251)    Pck.sub.-- AbGH + Pck.sub.-- tetRSv (4)                       female   29.125 (7.861)    Pck.sub.-- NbGH + Pck.sub.-- tetRsv (5)                       male     34.840 (14.745)    Pck.sub.-- NbGH + Pck.sub.-- tetRSv (4)                       female   28.125 (10.958)    Pck.sub.-- TbGH + Pck.sub.-- tetRSv (3)                       male     36.267 (11.402)    Pck.sub.-- TbGH + Pck.sub.-- tetRSv (2)                       female   27.300 (5.798)    NON-TRANSGENIC (6) male     29.583 (2.395)    NON-TRANSGENIC (6) female   23.117 (1.863)    ______________________________________

As expected for each co-injection, large animals, obviously expressingelevated levels of bGH, were observed as were animals of normal stature.

At 10 weeks of age, a sampling of transgenic female founders containingthe A+T and N+T were tested for induction of bGH in the serum using aradio-immune assay, after a single IP injection of 60 mg/kgtetracycline-HCl. The purpose of this experiment was simply to determinewhich if either of these two modified promoters was responsive torepression by tetR. The results are summarized in Table 4.

                  TABLE 4    ______________________________________    Construct           Animal  Weight  Basal   12 hours                                           36 hours    ______________________________________    249    2-5     21.1    0.00    0.00    0.00           female    250    6-6     42.9    4.6 ± 0.033                                   3.4 ± 0.062                                           4.9 ± 0.072           female    251    6-6     19.3    0.00    0.00    0.00           female    251    10-5    25.1    0.20 ± 0.008                                   0.19 ± 0.001                                           0.21 ± 0.038           female    252    5-2     38.7    0.59 ± 0.107                                   0.64 ± 0.044                                           1.12 ± 0.207           female    252    5-3     20.0    0.00    0.00    0.00           female    252    10-2    19.2    0.00    0.00    0.00    ______________________________________

No induction of bGH was observed in animals that lack the Pck₋₋ tetRSVgene (construct 250) or in animals with both the Pck₋₋ AbGH+Pck-tetRSvgenes (construct 251). An approximate two fold increase in serum bGHlevels was however detected in the 5-2 female which contains thePck-NbGH+Pck₋₋ tetRSV genes. The remainder of the animals hadundetectable levels of bGH expression, due in part to the relatively lowsensitivity of this assay. For example the 10-2 female (construct 252)shows no detectable bGH in the serum, but subsequent expieriments on heroffspring indicate that this line of animals does express bGH mRNA in atetracycline dependent manner. This initial data, suggested that thePck₋₋ N promoter was being regulated by tetR at least to a limitedextent.

To further characterize the mice, improve the sensitivity of the assayand to test the responsiveness of the Pck₋₋ T promoter, offspring offounder mice from each co-injection were produced. The transgenicprogeny were then raised in the presence or absence of tetracyclinemedicated water (500 μg/ml) for 4 weeks, prior to analysis of bGH mRNAexpression levels in the liver, the predominant site of PEPCKexpression. Northern blot hybridization analysis of these animals (FIG.7) demonstrated again, that animals with the Pck₋₋ NbGH gene wereresponsive to repression by tetR and that the other two modifiedpromoters exhibited no signs of tetR dependent regulation.

We attempted to breed all of the remaining founders containing thePck-NgGH+Pck₋₋ tetRSv genes to analyze their offspring in a similarmanner (FIG. 8). Of the 5 founders which produced offspring, 2 did notexpress bGH under any conditions, and from the remaining 3 onesegregated two different integration sites allowing us to establish atotal of 4 lines. All 4 lines exhibited tetracycline dependent bGHexpression as assayed by Northern blot hybridization. The efficiency oftetR repression appeared to be inversely correlated with the level ofexpression. For example 9-5 animals have the highest level of bGHexpression, show an obvious increase in body size, and exhibit onlymarginal tetR repression. In contrast 9-4Lc and 10-2 animals exhibitlower levels of tetracycline induced bGH expression, are of normalstature and appear to be efficiently regulated by tetR.

An S1 nuclease protection assay was performed to identify the start siteof transcription of bGH mRNA. As shown in FIG. 4, there was only onestart site identified regardless of the presence or absence of tetRrepressor binding. This start site was located approximately 20 bpdownstream from the TATA-box. At this location, the message isinitiating within the ∂∂7 operator sequence, just 3 or 4 base pairs 5'of the first tetR binding site.

7. EXAMPLE: OPTIMIZATION OF tetR CODING SEQUENCE

The use of the wild type Tn10 tetR gene in conjunction with the 252construct indicates that the TetR system can function in transgenicanimals and that in some cases, for instance in the 10-2 transgenicanimals, the level of regulation can be very high (FIGS. 9A and 9B).However, in other instances the efficiency of repression is not alwayscomplete, leading to a significant basal level of bGH expression. Thisfailure to repress may be due to low level expression of tetR. Tooptimize the expression of tetR repressor, a synthetic tetR gene wasgenerated which was devoid of splice signals and had optimized codonusage for mammalian cells.

7.1 Materials and Methods

7.1.1. TISSUE SPECIFICITY AND TETRACYCLINE INTRODUCTION OF bGH IN LINE10-2

For all Northern blots 10 μg of whole RNA was electrophoreses through a1% agarose gel containing 3% formaldehyde using standard techniques. Todetect bGH mRNA a random primed, radioactive bGH cDNA probe was used.All conditions for hybridization and washing of filters were done inaccordance with standard techniques of molecular biology.

7.1.2. EXPRESSION AND ALTERNATIVE PROCESSING OF THE tetR TRANSGENE

A RNase protection probe which extended from the NruI site of tetR 3' tothe end of the gene was used. This probe includes only tetR codingsequences and should give a fully protected fragment of approximately400 base pair. When hybridized to 150 μg of liver RNA (500,000 cpm ofprobe in a 30 μl hybridization consisting of 80% formamide, 40 mM PIPESpH 6.4, 400 mM NaOAc, and 1 mM EDTA), and digested with RNase one(Promega) for 30 minutes at 370 as recommended by the manufacturer, aprotected fragment of approximately 221-260 base pairs is observed, farsmaller than predicted.

7.1.3. 5' STRUCTURE OF tetR mRNA

Liver RNA was treated with reverse transcriptase and amplified by PCRusing the manufacturers recommended conditions (Pharmacia). The RNA wasamplified using two different pairs of primers. The first primer pair(TZ-1 and TZ-4) should produce a 619 base pair product. The secondprimer pair (TZ-3 and TZ-4) should produce a 498 base pair product. Thesequence of the primers are:

TZ-1: 5'CCGCATATGATCAATTCAAGGCCGAATAAG3'

TZ-3: 5'CTTTAGCGACTTGATGCTCTTGATCTTCCA3'

TZ-4: 5'AATTCGCCAGCCATGCCAAAAAAGAAGAGG3'

The TZ-4 primer is common to both primer pairs and is the 5' primerwhich encompasses the start codon of the tetR mRNA. Primer TZ-1 and TZ-3are two different 3' primers both of which are in the tetR codingregion. When amplified, these primer pairs produce smaller than expectedproducts (approx. 215 bp vs. 619 bp for TZ-4 and TZ-1, and approx. 94 bpvs. 498 bp for TZ-4 and TZ-3). The products of this reaction were clonedand sequenced. The sequence revealed the presence of an unexpectedintron which spanned from near the Xbal site at the start of tetR to asplice acceptor just 8 base pairs 5' of the TZ-3 primer.

7.1.4. 345 REPRESSOR CONSTRUCT

In an embodiment of the invention, any nuclear localization signal maybe added to a natural or synthetic tetR gene to facilitate itsexpression. For example, complementary oligonucleotides which encode anuclear localization signal sequence were synthesized (Oligos etc.) andadded in frame to the tetR coding sequences of pSTETR107 at the EcoR1and Xbal restriction sites to produce pNTETR. Oligonucleotide sequencesare: (GB1) (SEQ ID NO:24) 5'AATTCGCCAGCCATGCCAAAAAAGAAGAGGAAGGTAT3' and(GB2) (SEQ ID NO:24) 5'CTAGATACCTTCCTCTTCTTTTTTGGCATGGCTGGC3'. Whenannealed these oligonucleotides have a 5' EcoR1 and 3' Xbal compatibleoverhangs. These oligonucleotides fuse the amino acid sequence Met ProLys Lys Lys Arg, Lys Val,to the third amino acid (Arg) of wild typetetR.

Two complementary 51 base pair oligonucleotides which start the 5' capsite of bGH and extend to the first exon were synthesized (Oligos etc.).Sequence for the oligonucleotides are (5b-1) (SEQ ID NO:25):5'GATCCCAGGACCCAGTTCACCAGACGACTCAGGGTCCTGTGGACAGCT CAG3' and (5b-2) (SEQID NO:26): 5'AATTCTGAGCTGTCCACAGGACCCTGAGTCGTCTGGTGAACTGGGTCC TGG3'.When annealed these oligonucleotides have 5' BamH1 and 3'EcoR1compatible overhands. The oligonucleotides for the 5'leader sequence ofbGH were cloned into a BamH1, EcoR1 cut plasmid to produce p5' GH.

The nuclear localization modified tetR coding sequence was isolated bygel purification after restriction digestion of pNTETR using EcoR1 andHind III. This fragment was then inserted into p5'GH at the EcoR1 andHind III sites to product p5'GHTR.

To add the remainder of the bGH genomic sequence an intermediatemodification of p5'GHTR was first made. This modification consisted ofadding a Hind III--Pst1 linker to the Hind III site of p5'GHTR toproduct pGTO. The sequence of the oligonucleotides which comprise thislinker are: (CC-1) (SEQ ID NO:27) 5'AGCTTCTGCAG3' and (CC-2) (SEQ IDNO:28) 5'AGCTCTGCAGA3'. The remaining bGH genomic sequences were addedin two steps. First the Pst1 Sac2 fragment that begins in the first exonof bGH and ends in the third intron was excised from pSGH107. Similarly,the insert of pGTO which contains the 5' untranslated leader of bGH andthe nuclear localization modified tetR was excised using BamH1 and Pst1.These two gel purified fragments was then cloned into a BamH1 Sac2 cutvector to produce pGTG. Finally, the remainder of the bGH gene from theSac2 site in the third intron to the end of the gene, was added to pGTGby cutting pGTG with Sac2 and adding the Sac2 fragment from pSGH106 toproduce pNTETR-GH.

Plasmid pNTETR-GH was digested with BamH1 to excise the NTETR-GH gene.The fragment was cloned into the BamH1 site of pPCK 305 to produce thefinal plasmid pPCK-GHNTET. To produce transgenic mice, the PEPCK-GHTETgene was excised from the plasmid using Sa11 and Sac1. This fragment wasgel purified and coinjected with the PCK-NbGH gene previously describedto generate transgenic mice.

7.1.5. SYNTHETIC tetR COMPONENT SEQUENCES

The components of the synthetic tetR gene were synthesized by MidlandLaboratories as four overlapping double stranded DNA cassettes. Thesequence of these cassettes are shown in FIG. 15. Each cassette wasblunt cloned into the Hinc2 site of pUC19 and sequenced to verifyauthenticity. The resulting plasmids PLT1, pLT2, pLT3 and pLT5 can beused as the source material to assemble the entire synthetic tetR codingsequence since each contains an overlapping unique restriction site(bold face) through which they can be joined (pLT-1, pLT-2, pLT-3 andpLT-5 have been deposited with ATCC and have been assigned accessionnumbers 69396, 69397, 69398, and 69399respectively). There are manypossible ways by which these cassettes can be joined. By way of anexample, the inserts of plasmid pLT1 and pLT2 can be excised using EcoR1and Nsi1. The inserts can then be combined by cloning these twofragments into an EcoR1 vector. This procedure will assemble the 5' halfof the gene, using the overlapping Nsil restriction site to join thepieces. Similarly, the 3' half of the gene can be assembled from pLT3and pLT5 by cutting with EcoR1 and Sph1 (pLT3) and Sphl and Hind III(pLT5) to release the inserts. These inserts can then be joined at theoverlapping Sph1 site by cloning the fragments into an EcoR1, Hind IIIcut vector. Finally, the entire coding region can be put together usingthe overlapping restriction site ApaL1. This would result in a vectorwith the synthetic tetR coding sequence, as depicted in FIG. 16, clonedinto a plasmid as an EcoR1 Hind III fragment.

7.1.6. COMPOSITIONAL ANALYSIS OF WILD TYPE Tn10 tetR GENE

The Tn10 tetR coding sequence was analyzed on a desktop computer usingMac Vector software. FIG. 14 shows a diagram of the tetR coding regionwith all of the plus strand splice doner (D) and splice acceptor (A)signal sequences indicated. For reference the location of the Xbalrestriction is also indicated. The first graph depicts the percentage ofG and O bases in the coding region of tetR. There are several domains ofvery low GC content. The bottom graph is an analysis of codon bias. Thedark line is a comparison of the tetR codon usage to a mouse codon biastable. Values much lower than 1.0 are indicative of sequences which maytranslate poorly. For reference, a comparison of tetR to a Tobacco codonbias table is included (light line). In transgenic tobacco, the tetRregulation system functions very efficiently, suggesting that for thisgene, codon bias may be an important factor for efficient expression.

7.1.7. COMPOSITIONAL ANALYSIS OF SYNTHETIC tetR

FIG. 17 depicts the structure of the synthetic tetR gene, now devoid ofsplice donor signal sequences, with only a single splice acceptor signalremaining (A). This is not the splice acceptor which was active in the345 construct. The percentage of G and C bases has been significantlyimproved, while the frequency of CpG base pairs has been kept to aminimum. A CpG base pair is frequently the site for DNA methylation,which can negatively effect the expression of a gene. The codon bias ofthe synthetic tetR gene is vastly improved. The graph depicts theresults when the synthetic tetR coding sequence is compared to the samemouse codon bias table used previously.

7.2 Results

7.2.1. EXPRESSION OF tetR IN CONSTRUCT 345 OFFSPRING

To improve tetR expression a new repressor construct was produced. Theconstruct, referred to as Construct 345 is depicted in FIG. 10. In the345 construct the coding region of tetR is augmented with a nuclearlocalization signal sequence to increase the nuclear concentration ofrepressor. The tetR coding region was inserted into the first exon ofthe bGH gene. The bGH gene then acts as a genomic carrier, providingmultiple introns, which may improve expression, and a strongpolyadenylation signal, which may improve the processing and stabilityof the message.

The new repressor was coinjected with the bGH gene from construct 252.The resulting transgenic animals contain the new repressor, and a PEPCKregulated bGH gene with the tetR operators located just 3' of the PEPCKTATA-box element. Offspring of these animals were screened for bGHinduction (FIG. 11). Of the lines tested only one, line 14, showedtetracycline dependent regulation of bGH, and in this one case there wasstill a significant base level of bGH expression. Northern analysis,performed to determine the levels of tetR mRNA expressed in thetransgenic mice, indicated that the tetR gene was still not expressed ata high level.

To detect tetR mRNA with higher sensitivity the tetR mRNA was analyzedusing RNase protection. This technique revealed that the mRNA wasshorter then expected (FIG. 12). Subsequent analysis using reverseranscriptase-PCR with primers that amplify the entire coding region oftetR confirmed that the mRNA was significantly shorter then expected(FIG. 13). Sequence analysis of these RT-PCR products indicated that anunexpected splicing event had occurred. This splicing process occurredbetween a splice donor signal in the 5' end of the tetR coding regionand a splice acceptor approximately 400 bp 3' of the start codon. Theresulting mRNA is therefore deleted of the tetR DNA binding domain andabout two third of the entire coding region. This RNA could not possiblymake a functional repressor.

7.2.2. OPTIMIZATION OF tetR CONSTRUCT

A more detailed analysis of the tetR coding sequence indicated that thecodons used in this gene are poorly suited for expression in mammaliancells (FIG. 14). Therefore, it appears that the inefficiency of the tetRsystem is the result of two processes: (i) aberrant splicing of the RNAto produce a nonfunctional message; and (ii) inefficient translationwhich can lead to rapid mRNA turnover.

To circumvent the problems of internal splicing and potential problemsdue to codon bias and G-C content, a synthetic tetR gene was designed.The components of the synthetic tetR gene were synthesized as fouroverlapping double stranded cassettes. Each cassette was cloned inpuc19. The resulting plasmids designated pLT-1, pLT-2, pLT-3 and pLT-5,as depicted in FIG. 15, have been deposited with ATCC and assignedaccession numbers 69396, 69397, 69398, and 69399, respectively. Thesynthetic tetR (syn-tetR) has been designed to encode exactly the sameprotein product, but is devoid of splice signals and has greatlyimproved codon usage for mammalian cells. The sequence of the of thesyn-tetR is indicated in FIG. 16. The predicted analysis for splicingsignals, G+C content, and codon usage are depicted in FIG. 17.

8. DEPOSIT OF MICROORGANISMS

The following microorganisms have been deposited with the American TypeCulture Collection, (ATCC), Rockville, Md. and have been assigned thefollowing accession numbers:

    ______________________________________    Microorganism                 Date of Deposit                             Accession No.    ______________________________________    pLT-1        August 25, 1993                             69396    pLT-2        August 25, 1993                             69397    pLT-3        August 25, 1993                             69398    pLT-5        August 25, 1993                             69399    pPCK.sub.-- NbGH                 August 25, 1993                             69400    ______________________________________

The present invention is not to be limited in scope by themicroorganisms deposited since the deposited embodiments are intended asillustrations of single aspects of the invention and any microorganismswhich are functionally equivalent are within the scope of the invention.

The present invention is not to be limited in scope by the exemplifiedembodiments which are intended as illustrations of single aspects of theinvention, and any clones, DNA or amino acid sequences which arefunctionally equivalent are within the scope of the invention. Indeed,various modifications of the invention in addition to those describedherein will become apparent to those skilled in the art from theforegoing description and accompanying drawings. Such modifications areintended to fall within the scope of the appended claims.

It is also to be understood that all base pair sizes given fornucleotides are approximate and are used for purposes of description.

Various publications are cited herein, which are hereby incorporated byreference in their entirety.

    __________________________________________________________________________    #             SEQUENCE LISTING    - (1) GENERAL INFORMATION:    -    (iii) NUMBER OF SEQUENCES: 29    - (2) INFORMATION FOR SEQ ID NO:1:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 59 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: unknown    -     (ii) MOLECULE TYPE: DNA (genomic)    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    - TTGACACTCT ATCATTGATA GAGTTATTTT ACCACTCCCT ATCAGTGATA GA - #GAAAAGT      59    - (2) INFORMATION FOR SEQ ID NO:2:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 70 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: unknown    -     (ii) MOLECULE TYPE: DNA (genomic)    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    - GAATTCGATA CTCTATCATT GATAGAGTAT CAAGCTTATC CCTATCAGTG AT - #AGAGATAC      60    #        70    - (2) INFORMATION FOR SEQ ID NO:3:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 49 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: unknown    -     (ii) MOLECULE TYPE: DNA (genomic)    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    #               49GAGTT ACTATTTAAA TCCCTATCAG TGATAGAGA    - (2) INFORMATION FOR SEQ ID NO:4:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 71 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: unknown    -     (ii) MOLECULE TYPE: DNA (genomic)    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    - GGAATTCGAT ACTCTATCAT TGATAGAGTA TCAAGCTTAT CCCTATCAGT GA - #TAGAGATA      60    #       71    - (2) INFORMATION FOR SEQ ID NO:5:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 624 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: unknown    -     (ii) MOLECULE TYPE: DNA (genomic)    -     (ix) FEATURE:              (A) NAME/KEY: CDS              (B) LOCATION: 1..624    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:    - ATG TCT AGA TTA GAT AAA AGT AAA GTG ATT AA - #C AGC GCA TTA GAG CTG      48    Met Ser Arg Leu Asp Lys Ser Lys Val Ile As - #n Ser Ala Leu Glu Leu    #                 15    - CTT AAT GAG GTC GGA ATC GAA GGT TTA ACA AC - #C CGT AAA CTC GCC CAG      96    Leu Asn Glu Val Gly Ile Glu Gly Leu Thr Th - #r Arg Lys Leu Ala Gln    #             30    - AAG CTA GGT GTA GAG CAG CCT ACA TTG TAT TG - #G CAT GTA AAA AAT AAG     144    Lys Leu Gly Val Glu Gln Pro Thr Leu Tyr Tr - #p His Val Lys Asn Lys    #         45    - CGG GCT TTG CTC GAC GCC TTA GCC ATT GAG AT - #G TTA GAT AGG CAC CAT     192    Arg Ala Leu Leu Asp Ala Leu Ala Ile Glu Me - #t Leu Asp Arg His His    #     60    - ACT CAC TTT TGC CCT TTA GAA GGG GAA AGC TG - #G CAA GAT TTT TTA CGT     240    Thr His Phe Cys Pro Leu Glu Gly Glu Ser Tr - #p Gln Asp Phe Leu Arg    # 80    - AAT AAC GCT AAA AGT TTT AGA TGT GCT TTA CT - #A AGT CAT CGC GAT GGA     288    Asn Asn Ala Lys Ser Phe Arg Cys Ala Leu Le - #u Ser His Arg Asp Gly    #                 95    - GCA AAA GTA CAT TTA GGT ACA CGG CCT ACA GA - #A AAA CAG TAT GAA ACT     336    Ala Lys Val His Leu Gly Thr Arg Pro Thr Gl - #u Lys Gln Tyr Glu Thr    #           110    - CTC GAA AAT CAA TTA GCC TTT TTA TGC CAA CA - #A GGT TTT TCA CTA GAG     384    Leu Glu Asn Gln Leu Ala Phe Leu Cys Gln Gl - #n Gly Phe Ser Leu Glu    #       125    - AAT GCA TTA TAT GCA CTC AGC GCT GTG GGG CA - #T TTT ACT TTA GGT TGC     432    Asn Ala Leu Tyr Ala Leu Ser Ala Val Gly Hi - #s Phe Thr Leu Gly Cys    #   140    - GTA TTG GAA GAT CAA GAG CAT CAA GTC GCT AA - #A GAA GAA AGG GAA ACA     480    Val Leu Glu Asp Gln Glu His Gln Val Ala Ly - #s Glu Glu Arg Glu Thr    145                 1 - #50                 1 - #55                 1 -    #60    - CCT ACT ACT GAT AGT ATG CCG CCA TTA TTA CG - #A CAA GCT ATC GAA TTA     528    Pro Thr Thr Asp Ser Met Pro Pro Leu Leu Ar - #g Gln Ala Ile Glu Leu    #               175    - TTT GAT CAC CAA GGT GCA GAG CCA GCC TTC TT - #A TTC GGC CTT GAA TTG     576    Phe Asp His Gln Gly Ala Glu Pro Ala Phe Le - #u Phe Gly Leu Glu Leu    #           190    - ATC ATA TGC GGA TTA GAA AAA CAA CTT AAA TG - #T GAA AGT GGG TCT TAA     624    Ile Ile Cys Gly Leu Glu Lys Gln Leu Lys Cy - #s Glu Ser Gly Ser  *    #       205    - (2) INFORMATION FOR SEQ ID NO:6:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  208 ami - #no acids              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:    - Met Ser Arg Leu Asp Lys Ser Lys Val Ile As - #n Ser Ala Leu Glu Leu    #                 15    - Leu Asn Glu Val Gly Ile Glu Gly Leu Thr Th - #r Arg Lys Leu Ala Gln    #             30    - Lys Leu Gly Val Glu Gln Pro Thr Leu Tyr Tr - #p His Val Lys Asn Lys    #         45    - Arg Ala Leu Leu Asp Ala Leu Ala Ile Glu Me - #t Leu Asp Arg His His    #     60    - Thr His Phe Cys Pro Leu Glu Gly Glu Ser Tr - #p Gln Asp Phe Leu Arg    # 80    - Asn Asn Ala Lys Ser Phe Arg Cys Ala Leu Le - #u Ser His Arg Asp Gly    #                 95    - Ala Lys Val His Leu Gly Thr Arg Pro Thr Gl - #u Lys Gln Tyr Glu Thr    #           110    - Leu Glu Asn Gln Leu Ala Phe Leu Cys Gln Gl - #n Gly Phe Ser Leu Glu    #       125    - Asn Ala Leu Tyr Ala Leu Ser Ala Val Gly Hi - #s Phe Thr Leu Gly Cys    #   140    - Val Leu Glu Asp Gln Glu His Gln Val Ala Ly - #s Glu Glu Arg Glu Thr    145                 1 - #50                 1 - #55                 1 -    #60    - Pro Thr Thr Asp Ser Met Pro Pro Leu Leu Ar - #g Gln Ala Ile Glu Leu    #               175    - Phe Asp His Gln Gly Ala Glu Pro Ala Phe Le - #u Phe Gly Leu Glu Leu    #           190    - Ile Ile Cys Gly Leu Glu Lys Gln Leu Lys Cy - #s Glu Ser Gly Ser    #       205    - (2) INFORMATION FOR SEQ ID NO:7:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 92 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: unknown    -     (ii) MOLECULE TYPE: DNA (genomic)    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:    - CGGCCCTATA AAAAGCGAAG CGCGCGGCGG GCGGGAGTCG CTGCGTTGCC TT - #CGCCCCGT      60    #          92      CCTC GCGCCGCCCG CC    - (2) INFORMATION FOR SEQ ID NO:8:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 61 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: unknown    -     (ii) MOLECULE TYPE: DNA (genomic)    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:    - AAGAAGTATA TTAGAGCGAG TCTTTCTGCA CACACGATCA CCTTTCCTAT CA - #ACCCCACT      60    #               61    - (2) INFORMATION FOR SEQ ID NO:9:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 74 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: unknown    -     (ii) MOLECULE TYPE: DNA (genomic)    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:    - GTATTATGTT TTATGTTACT GTAAAAGATG TAAAGAGAGG CACGTGGTTA AG - #CTCTCGGG      60    #     74    - (2) INFORMATION FOR SEQ ID NO:10:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 73 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: unknown    -     (ii) MOLECULE TYPE: DNA (genomic)    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:    - CGCCCCAAGC ATAAACCCTG GCGCGCTCGC GGCCCGGCAC TCTTCTGGTC CC - #CACAGACT      60    #      73    - (2) INFORMATION FOR SEQ ID NO:11:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 74 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: unknown    -     (ii) MOLECULE TYPE: DNA (genomic)    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:    - TAGGCAGCAG GCATATGGGA TGGGATATAA AGGGGCTGGA GCACTGAGAG CT - #GTCAGAGA      60    #     74    - (2) INFORMATION FOR SEQ ID NO:12:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 19 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: unknown    -     (ii) MOLECULE TYPE: DNA (genomic)    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:    # 19               AGT    - (2) INFORMATION FOR SEQ ID NO:13:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 19 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: unknown    -     (ii) MOLECULE TYPE: DNA (genomic)    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:    # 19               AGT    - (2) INFORMATION FOR SEQ ID NO:14:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 19 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: unknown    -     (ii) MOLECULE TYPE: DNA (genomic)    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:    # 19               AGA    - (2) INFORMATION FOR SEQ ID NO:15:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 19 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: unknown    -     (ii) MOLECULE TYPE: DNA (genomic)    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:    # 19               GGA    - (2) INFORMATION FOR SEQ ID NO:16:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 189 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: unknown    -     (ii) MOLECULE TYPE: DNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:    - GATATCGAAT TCATGAGTAG ATTGGACAAG AGCAAAGTGA TCAATAGTGC TC - #TGGAGCTG      60    - TTGAATGAAG TGGGCATAGA AGGTCTGACT ACCAGAAAGC TGGCCCAGAA GC - #TGGGAGTG     120    - GAGCAGCCAA CATTGTACTG GCATGTGAAG AATAAGAGGG CTCTGCTGGA TG - #CATTGGCG     180    #        189    - (2) INFORMATION FOR SEQ ID NO:17:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 169 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: unknown    -     (ii) MOLECULE TYPE: DNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:    - GCTCGGTACC TGGATGCATT GGCCATTGAG ATGCTGGACA GACACCATAC AC - #ACTTCTGC      60    - CCACTGGAAG GCGAGAGTTG GCAGGACTTC CTGAGGAACA ATGCTAAGAG TT - #TCAGATGT     120    #              169AGAGA CGGTGCTAAA GTGCACCTGG AATTCGAGC    - (2) INFORMATION FOR SEQ ID NO:18:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 231 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: unknown    -     (ii) MOLECULE TYPE: DNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:    - GCTCGAATTC AAAGTGCACC TGGGTACAAG GCCAACAGAG AAACAGTACG AG - #ACCCTGGA      60    - GAACCAGCTG GCATTTCTGT GCCAACAAGG CTTCAGCCTG GAGAATGCAT TG - #TATGCTCT     120    - GAGTGCTGTG GGTCACTTCA CACTGGGTTG TCTCCTGGAG GACCAGGAGC AC - #CAGGTGGC     180    #            231GAGACCC CAACCACTGA CAGCATGCCC CGGATCCGAG C    - (2) INFORMATION FOR SEQ ID NO:19:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 155 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: unknown    -     (ii) MOLECULE TYPE: DNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:    - GCTCGGATCC ACAGCATGCC CCCATTGCTG AGACAGGCCT ATGAGCTGTT TG - #ACCACCAA      60    - GGGGCAGAGC CTGCTTTTCT GTTTGGCCTG GAGCTCATCA TCTGTGGTCT GG - #AGAAGCAG     120    #      155         GCTC CTGAAGCTTG ATATC    - (2) INFORMATION FOR SEQ ID NO:20:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 647 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: unknown    -     (ii) MOLECULE TYPE: DNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:    - GATATCGAAT TCATGAGTAG ATTGGACAAG AGCAAAGTGA TCAATAGTGC TC - #TGGAGCTG      60    - TTGAATGAAG TGGGCATAGA AGGTCTGACT ACCAGAAAGC TGGCCCAGAA GC - #TGGGAGTG     120    - GAGCAGCCAA CATTGTACTG GCATGTGAAG AATAAGAGGG CTCTGCTGGA TG - #CATTGGCC     180    - ATTGAGATGC TGGACAGACA CCATACACAC TTCTGCCCAC TGGAAGGCGA GA - #GTTGGCAG     240    - GACTTCCTGA GGAACAATGC TAAGAGTTTC AGATGTGCTC TGTTGAGCCA CA - #GAGACGGT     300    - GCTAAAGTGC ACCTGGGTAC AAGGCCAACA GAGAAACAGT ACGAGACCCT GG - #AGAACCAG     360    - CTGGCATTTC TGTGCCAACA AGGCTTCAGC CTGGAGAATG CATTGTATGC TC - #TGAGTGCT     420    - GTGGGTCACT TCACACTGGG TTGTGTCCTG GAGGACCAGG AGCACCAGGT GG - #CCAAGGAG     480    - GAGAGGGAGA CCCCAACCAC TGACAGCATG CCCCCATTGC TGAGACAGGC CA - #TAGAGCTG     540    - TTTGACCACC AAGGGGCAGA GCCTGCTTTT CTGTTTGGCC TGGAGCTCAT CA - #TCTGTGGT     600    #               647AGTG TGAGAGTGGC TCCTGAAGCT TGATATC    - (2) INFORMATION FOR SEQ ID NO:21:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 30 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: unknown    -     (ii) MOLECULE TYPE: DNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:    #           30     CAAG GCCGAATAAG    - (2) INFORMATION FOR SEQ ID NO:22:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 30 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: unknown    -     (ii) MOLECULE TYPE: DNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:    #           30     CTCT TGATCTTCCA    - (2) INFORMATION FOR SEQ ID NO:23:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 30 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: unknown    -     (ii) MOLECULE TYPE: DNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:    #           30     CAAA AAAGAAGAGG    - (2) INFORMATION FOR SEQ ID NO:24:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 37 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: unknown    -     (ii) MOLECULE TYPE: DNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:    #      37          CAAA AAAGAAGAGG AAGGTAT    - (2) INFORMATION FOR SEQ ID NO:25:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 51 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: unknown    -     (ii) MOLECULE TYPE: DNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:    #             51AGTTCAC CAGACGACTC AGGGTCCTGT GGACAGCTCA G    - (2) INFORMATION FOR SEQ ID NO:26:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 51 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: unknown    -     (ii) MOLECULE TYPE: DNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:    #             51CCACAGG ACCCTGAGTC GTCTGGTGAA CTGGGTCCTG G    - (2) INFORMATION FOR SEQ ID NO:27:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 11 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: unknown    -     (ii) MOLECULE TYPE: DNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:    #       11    - (2) INFORMATION FOR SEQ ID NO:28:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 11 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: unknown    -     (ii) MOLECULE TYPE: DNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:    #       11    - (2) INFORMATION FOR SEQ ID NO:29:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 36 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: unknown    -     (ii) MOLECULE TYPE: DNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:    #       36         TCTT TTTTGGCATG GCTGGC    __________________________________________________________________________     ##SPC1##

What is claimed is:
 1. A purified and isolated nucleic acid moleculecomprising an optimized tetracycline repressor (tetR) gene as depictedin FIG. 16 (SEQ ID NO: 20).
 2. A purified and isolated nucleic acidmolecule comprising an optimized tetR gene wherein the tetR gene isdevoid of any splice signals and is modified to optimize codon usage formammalian cells and to increase the percentage of G and C bases whilemaintaining a low frequency of CpG base pairs.
 3. An isolated mammalianhost cell which contains and expresses the nucleic acid molecule ofclaim
 2. 4. An isolated mammalian host cell which contains and expressesan optimized tetR gene as depicted in FIG. 16 (SEQ ID NO: 20).
 5. Amethod for expressing the tetR gene in mammalian cells in vitro, whereinthe tetR gene is devoid of any splice signals and is modified tooptimize codon usage for mammalian cells and to increase the percentageof G and C bases while maintaining a low frequency of CpG base pairs,said method comprising introducing said tetR gene to said mammaliancells, and culturing said mammalian cells under conditions sufficientfor the expression of said tetR gene.
 6. A transgenic mouse having atransgene integrated into its genome, wherein the transgene comprises anoptimized tetR gene having a sequence set forth in FIG. 16 (SEQ ID NO:20) in operable linkage with an unmodified PEPCK promoter, and whereinsaid transgene is expressed in the cells of said mouse at a levelsufficient to produce the optimized tetR protein.
 7. A transgenic mouseaccording to claim 6 further having a second transgene integrated intoits genome, wherein said second transgene comprises a gene of interestin operable linkage with a PEPCK promoter element modified to contain atetR operator at the NheI site, wherein in the absence of a tetracyclinecompound, expression of said gene of interest is repressed in the cellsof said mouse, and in the presence of a tetracycline compound in themouse, said gene of interest is expressed in the cells of the mousecausing said mouse to exhibit a detectable and functional phenotype ascompared to a wild-type mouse.
 8. A method of selectively inducing theexpression of a gene of interest in a transgenic mouse comprisingadministering a tetracycline compound to the transgenic mouse of claim7, wherein said tetracycline compound induces the expression of saidgene of interest.
 9. A transgenic mouse having a transgene integratedinto its genome, wherein the transgene comprises the optimized tetR geneof claim 2 in operable linkage with an unmodified PEPCK promoter,wherein said transgene is expressed in the cells of said mouse at alevel sufficient to produce the optimized tetR protein in an amounteffective to regulate expression of a gene of interest upon itsintroduction into said mouse at an embryonic stage, and wherein saidgene of interest is in operable linkage with a PEPCK promoter elementmodified to contain a tetR operator at the NheI site.
 10. A transgenicmouse according to claim 9 further having a second transgene integratedinto its genome, wherein said second transgene comprises a gene ofinterest in operable linkage with a PEPCK promoter element modified tocontain a tetR operator at the NheI site, wherein in the absence of atetracycline compound, expression of said gene of interest is repressedin the cells of said mouse, and in the presence of a tetracyclinecompound, said gene of interest is expressed in the cells of the mousecausing said mouse to exhibit a detectable and functional phenotype ascompared to a wild-type mouse.
 11. The transgenic mouse of claim 10,wherein said gene of interest is the gene encoding bovine growthhormone, and wherein in the absence of a tetracycline compound, theexpression of bovine growth hormone is repressed, and in the presence ofa tetracycline compound, said gene encoding bovine growth hormone isexpressed in the cells of the mouse causing said mouse to exhibitaccelerated growth as compared to a wild-type mouse.
 12. A method ofselectively regulating the expression of a gene of interest in atransgenic mouse comprising administering tetracycline, or functionallyrelated compound, to the transgenic mouse of claim 11, wherein saidtetracycline, or functionally related compound, regulates the expressionof said gene of interest.